Plants can be damaged by a wide variety of pathogenic organisms including viruses, bacteria, fungi and nematodes. Annual crop losses due to these pathogens is in the billions of dollars. Synthetic pesticides represent one form of defense against pathogens, and each year thousands of tons of such chemicals are applied to farm land and agricultural commodities. The cost of chemical pesticides is measured not only in the cost of producing these pesticides but also in both short term and long term environmental damage and the inherent risks to human health.
Plants also contain their own innate mechanisms of defense against pathogenic organisms. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and bred into many crop plants. These natural disease resistance genes often provide high levels of resistance (or immunity) to pathogens and represent the most economical and environmentally friendly form of crop protection. Despite the commercial significance, little is known about the molecular basis of natural disease resistance.
It has been postulated that disease resistance may be induced by the interaction of single genes in both the pathogen and the plant host. See A. H. Flor, "Host-Parasite Interactions in Flat-Rust--its Genetics and Other Implications," Phytopath, 45:680-685 (1947) and A. H. Flor, "Current Status of the Gene-for-Gene Concept," Ann. Rev. Phytopath, 9:275-96 (1971), both of which are hereby incorporated by reference. Many plant disease resistance genes have been mapped to single loci, and individual avirulence genes have been isolated from bacterial and fungal pathogens. See B. Staskawicz, et al., "Cloned Avirulence Gene of Pseudomonas syringae pv. glycinea Determines Race-Specific Incompatibility on Glycine max Lo, Merr." Proc. Natl. Acad. Sci USA," 81:6024-28 (1984); A. M. Ellingboe, "Genetics of Host-Parasite Interactions," Encyclopedia of Plant Pathology, New Series, Vol. 4: Physiological Plant Pathology, pp. 761-78 (1976); D. W. Gabriel et al. "Gene-for-Gene Interactions of Five Cloned Avirulence Genes from Xanthomonas campestris pv malvacearum with Specific Resistance Genes in Cotton," Proc Natl Acad Sci USA 83:6415-19 (1986); S. H. Hulbert, et al., "Recombination at the Rp1 Locus of Maize," Mol. Gen. Genet., 226:377-82 (1991); N. T. Keen, et al., "New Disease Resistance Genes in Soybean Against Pseudomonas syringae pv. glycinea: Evidence That One of Them Interacts with a Bacterial Elicitor," Theor. Appl. Genet. 81:133-38 (1991); R. Messeguer et al., "High Resolution RFLP Map Around the Root-knot Nematode Resistance Gene (Mi) in Tomato," Theor. Appl. Genet 82:529-3G (1991); T. Debener, et al., "Identification and Molecular Mapping of a Single Arabidopsis thaliana locus Determining Resistance to a Phytopathogenic Pseudomonas syringae isolate," Plant J 1:289:302 (1991); D. Y. Kobayashi, et al. "Cloned Avirulence Genes from Tomato Pathogen Pseudomonas syringae pv. tomato Confer Cultivar Specificity on Soybean," Proc. Natl. Acad. Sci. USA 86:157-61 (1989); and J. A. L. Van Kan, et al., "Cloning and Characterization of cDNA of Avirulence Gene avr9 of the Fungal Pathogen Cladosporium fulvum, Causal Agent of Tomato Leaf Mold," Mol. Plant-Microbe Interactions 4:52-59 (1991), all of which are hereby incorporated by reference. However, despite this progress, the molecular isolation of plant disease resistance genes has been hindered by the fact that little is known of the gene products encoded at these loci.
The phenomenon of disease resistance is believed to be initiated by physical contact between a pathogen and a potentially compatible portion of the host. Once such contact has occurred, usually as a result of wind or rain vectored deposition of the pathogen, the pathogen must recognize that such contact has been established in order to initiate the pathogenic process. Likewise, such recognition by the host is required in order to initiate a resistance response. The precise manner in which such recognition occurs is not clear. However, pathogen recognition is believed to be associated with low pH of plant tissues or the presence of plant-specific metabolites. On the other hand, recognition by the host involves at least two partly separate pathways of recognition. A general mechanism detects a presence of the cell wall fragments from the pathogen and/or the damaged host. In addition, recognition results from a race-specific mechanism where the host disease resistance gene recognizes the avirulence gene of the pathogen. Both host recognition mechanisms lead to one or more levels of gene activation which in turn lead to production of defensive resistance factors (e.g., gum or cork production, production of inhibitors of pathogen proteases, deposition of lignin and hydroxyproplin-rich proteins in cell walls) and offensive resistance factors (e.g., production of phytoalexins, secreted chitinases). If the rate and level of activation of the genes producing these factors is sufficiently high, the host is able to gain an advantage on the pathogen. On the other hand, if the pathogen is fully activated at an earlier stage in the infection process, it may overwhelm both the offensive and defensive resistance factors of the plant. The phenomenon of disease resistance is fully discussed in J. L. Bennetzen et al., "Approaches and Progress in the Molecular Cloning of Plant Disease Resistance Genes," Genetic Engineering, 14:99-124 (1992), which is hereby incorporated by reference.
Recently, elicitors of plant defense responses have been shown to induce phosphorylation and dephosphorylation of specific plant proteins, and inhibitors of mammalian protein kinases were found to inhibit expression of certain plant defense genes. See G. Felix, et al., "Rapid Changes of Protein Phosphorylation are Involved in Transduction of the Elicitor Signal in Plant Cells," Proc. Natl. Acad. Sci. USA, 88:8831-34 (1991); V. Raz, et al., "Ethylene Signal is Transduced via Protein Phosphorylation Events in Plants," The Plant Cell, 5:523-30 (1993); and E. E. Farmer, et al. "Oligosaccharide Signaling in Plants--Specificity of Oligouronide-Enhanced Plasma Membrane Protein Phosphorylation," J. Biological Chemistry, 266:3140-45 (1991), all of which are hereby incorporated by reference. At best, these references suggest that kinases are present in the metabolic pathway of disease resistance. These publications, however, do not disclose a gene which confers disease resistance to plants by responding to an avirulence gene in plant pathogens.
In tomato, resistance to the bacterial pathogen Pseudomonas syringae pv. tomato is encoded by a single locus (Pto) that displays dominant gene action. See R. E. Pitbaldo et al., "Genetic Basis of Resistance to Pseudomonas syringae pv. tomato in Field Tomatoes," Can. J. Plant Path., 5:251-55 (1983) ("Pitbaldo 1983"), which is hereby incorporated by reference. As with many commercially important traits in cultivated tomato (Lycopersicon esculentum), the resistance was identified in a wild tomato species, specifically Lycopersicon pimpinellifolium. See Pitbaldo 1983. Since the Pto gene was introgressed into tomato from a wild species, the region around the locus is polymorphic with respect to L. esculentum DNA. This polymorphism has been exploited by using a strategy relying on near-isogenic lines to identify molecular markers closely linked to Pto. See G. B. Martin, et al., "Rapid Identification of Markers Linked to Pseudomonas Resistance Gene in Tomato Using Random Primers and Near-isogenic Lines," Proc. Natl. Acad. Sci. USA, 88:2336-40 (1991) ("Martin et. al. 1991"), which is hereby incorporated by reference. Significant effort has been undertaken to map genetically the Pto gene. See G. B. Martin, et al. "High Resolution Linkage Analysis and Physical Characterization of the Pto Bacterial Resistance in Tomato," Molecular Plant Microbe Interaction, 6:21-34 (1993) ("Martin et. al. 1993") and G. B. Martin, et al, "Towards Positional Cloning of the Pto Bacterial Resistance Locus From Tomato," Advances in Molecular Genetics of Plant-Microbe Interactions, pp. 451-55 (1993). Moreover, the Pto gene is present in a number of commercial tomato varieties where it provides complete protection against Pseudomonas syringae pv. tomato bacteria and the disease referred to as "bacterial specks". Despite its wide-spread commercial use, no one has cloned or molecularly analyzed/characterized the Pto gene from tomato or a related disease resistance gene from any other plant species.